Recently several G protein-coupled receptors (GPCRs) have been shown to localize to intracellular membranes, in particular the nuclear membrane. As such, we sought to determine if the β-adrenergic receptor (βAR) subtypes and their associated signalling machinery are functionally localized to nuclear membranes. We demonstrated the presence of β1AR and the β3AR, but not the β2AR, in adult ventricular myocyte nuclei by western blotting, confocal microscopy and functional assays. Downstream signalling partners such as GαS, Gαi and adenylyl cyclase II and V/VI were also present. Nuclear-localized βARs were functional with respect to ligand binding and effector activation. In isolated nuclei, the non-selective βAR agonist isoproterenol (ISO) and the β3AR-selective ligand BRL37344, but not the β1AR-selective xamoterol, stimulated
transcription initiation in a pertussis toxin (PTX)-sensitive manner. In contrast, stimulation of type B endothelin receptors (ETB), another GPCR family shown to be
present on the nuclear membrane, decreased de novo RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the
presence or absence of inhibitors of the PI3K/PKB and mitogen-activated protein kinase
(MAPK) pathways. Components of p38, JNK, and ERK1/2 MAPK cascades as well as
PKB were detected in nuclear preparations. Inhibition of PKB with triciribine converted
the activation of the βAR from stimulatory to inhibitory with regards to transcription
initiation. Analysis by qPCR indicated isoproterenol treatment increased 18S rRNA but
decreased NFκB mRNA. In contrast, ET-1 had no effect on 18S rRNA expression.
Further investigation using pathway-specific PCR arrays revealed that isoproterenol
treatment also reduced the expression of several other genes involved in the activation
of NFκB and that ERK1/2 and PKB inhibitors attenuated this effect. Subsequent
genome-wide microarray analysis has revealed that nuclear βAR and ETB regulated a
host of genes in an overlapping but distinct manner. Moreover, both ET-1 and ISO
produced an L-NAME-sensitive increase in NO production in isolated cardiac nuclei.
These observations were confirmed in intact cardiomyocytes using novel caged
analogues of ISO and ET-1 and the cell-permable NO-sensitive fluorescent dye, DAF-2
DA. Briefly, both ET-1 and isoproterenol increased NO production, and this increase
was prevented upon preincubation with L-NAME. Moreover, the ability of isoproterenol
to increase transcription initiation in isolated nuclei was blocked by L-NAME or the
PKG inhibitor KT5823, indicating the NO-GC-PKG pathway is involved in the
regulation of gene expression by nuclear βARs. Hence, we have shown that βARs and
ETRs in the nuclear membrane activate distinct signalling pathways, resulting in
different effects on gene transcription and thus represent potentially important targets
for drug development.